Effects Of L-655,708 On Learning And Memory Of Normal Rats By Multi-level Analysis

【Purpose】At present,whetherα5 subunit containing GABA_Areceptors(α5-GABA_ARs)reverse agonists can improve learning and memory of normal animals is greatly inconsistent.It has been attributed to three main factors:different selectivity of drugs for receptors,duration of effective plasma concentration and variable effects in different stages of memory.In this study,a high binding selectivityα5-GABA_ARs reverse agonist,L-655,708,was selected as a representative to be explored.A low-multiple dose regimen based on its half-life was designed,to maintain effective plasma concentration over a relatively long period.The dose regimen was administered in three different periods of learning and memory(before-learning,during-learning and before-recalling),and its effects on learning and memory of normal rats were evaluated.【Subjects and Methods】In this study,male SD rats weighing 270-300 g were randomly assigned to different groups according to the demand quantity of different experiments.The study was carried out in three parts.In the first part,by high performance liquid chromatography(HPLC)analysis,the changes of plasma concentration in rats after a single intraperitoneal injection of L-655,708 were detected,and the half-life of L-655,708 was calculated.Based on its half-life,a low-multiple dose regimen was designed,and its plasma concentration after administration was further explored.Then,to clarify whether the dose regimen could lead to excessively binding to other subunits except forα5 subunits containing GABA_ARs due to high plasma concentration to cause side effects,elevated plus maze test(EPM)was used to evaluate anxiety of rats.Finally,the safety of L-655,708 used in vivo was verified by blood routine detection and H&E staining.In the second part,the designed low-multiple dose regimen was applied to three different periods(before-learning,during-learning and before-recalling)of Morris water maze(MWM)test,to make it affect different stages of memory process,such as information acquisition,consolidation and recall.Its effects on cognitive function of normal rats were evaluated by MWM test,and compared with high-single dose regimen commonly used in previous behavioral studies and vehicle control.Then,changes at molecular level were detected by western blot analysis of hippocampal tissues.Levels of these molecules,including GABA_Areceptors,glutamate receptors,intracellular signaling molecules and nuclear transcription factors were detected,which were closely related to hippocampus-dependent memory,and could provide powerful evidence for behavioral changes.In the third part,the experimental procedure that improved memory of normal rats in the second part was adopted.After short-term intensive learning of MWM test,resting-state functional magnetic resonance imaging(f MRI)data of low-multiple dose group,vehicle control group and learning control group were collected by 9.4 T ultra-high field magnetic resonance imaging system.Then,we explored the changes of neural activity in different hippocampal subregions by mean amplitude of low-frequency fluctuation(m ALFF)analysis.The functional connectivity(FC)was assessed by using region of interest(ROI)-based analysis between different hippocampal subregions and cortical regions,to complement and support the findings of MWM behavior test and Western blot analysis.【Results】1.We found that the half-life ofα5-GABA_ARs reverse agonist L-655,708 was about 30 min by single intraperitoneal injection.The low-multiple dose regimen designed based on its half-life,could maintain effective plasma concentration over a relatively long period,and prolong the duration according to requirements of different tests by increasing administration times.Moreover,the low-multiple dose regimen still maintained high selectivity forα5 subunit containing GABA_Areceptors and had no anxiety side effects confirmed by EPM test.In addition,the dose regimen did not cause toxic effects to be used in vivo indicated by blood routine detection and H&E staining.2.MWM test and Western blot analysis showed that L-655,708 administration in different periods under different dose regimens,had different effects on learning and memory of normal rats,and caused different changes at the molecular level.When L-655,708 was administered in before-learning and before-recalling period respectively,neither the signaling molecules nor cognitive function showed significant changes.When L-655,708 was administered in during-learning period,the low-multiple dose regimen improved spatial memory of normal rats,while the high-single dose regimen commonly used in previous behavioral studies could not.Meanwhile,the levels of glutamate receptors(NMDAR1),phosphorylated intracellular signaling molecules(p-PKA and p-Ca MKII)and nuclear transcription factors(p-CREB-1)were also changed,which provided powerful support for functional changes.The results confirmed that L-655,708 could improve spatial memory of normal rats,and its effects were affected by the duration of effective plasma concentration and time periods of administration.3.By m ALFF analysis,we found that,after intensive learning of MWM test,the neural activity of different hippocampal subregions was increased,and it was more obvious under L-655,708 administration.The FCs between hippocampal subregions and the medial prefrontal cortex,cingulate cortex and motor cortex was enhanced under the intensive learning.Moreover,under L-655,708 administration in during-learning period,the increased FC between hippocampus and cingulate cortex was more obvious,and the FC between hippocampus and retosplenial cortex,motor cortex and somatosensory cortex also showed a trend towards increase.Additionally,the changes of neural activity in hippocampus,and the FC between hippocampus and cingulate cortex were significantly correlated with the learning performance of rats.These findings suggest that L-655,708,aα5-GABA_ARs reverse agonist,could obviously increase the neural activity of hippocampus and the FC between hippocampus and cortex,thus promoting learning and memory of normal rats.【Conclusion】In this study,to explore the effects ofα5-GABA_ARs reverse agonist on cognitive function of normal rats,L-655,708 with high selectivity forα5-GABA_ARs was selected as a representative,and a low-multiple dose regimen was designed to maintain effective plasma concentration much longer.Combining Morris water maze behavior test,signaling molecules detection by Western blot and resting state f MRI study,this study confirmed thatα5-GABA_ARs reverse agonists represented by L-655,708 could improve cognitive function of normal rats,which further strengthened the awareness of their effects on cognitive function.In addition,this study also showed that effective plasma concentration maintenance and dose administration on different stages of memory did affect behavioral effects,to which should be paid more attention when exploring the effects of similar drugs in future,and the designed research procedure also had important significance for reference.